Gene to protein Flashcards
What is the centra dogma?
1 - DNA is heritable material used to store and transmit biological information from generation to generation
2 - RNA is a messenger to transfer info in DNA into cell to make proteins
3 - proteins carry out cellular functions
What are the 2 processes involved in gene expression?
Transcription and translation
What are the four levels of gene expression control before protein expression is even started? Where are these controls in the cell?
Transcription, RNA processing (in the nucleus), Nuclear export (nucleus membrane pores) and translation (in the ribosomes)
Why is control of gene expression important? Give and example
Housekeeping proteins are always needed in large quantities (e.g. Tubulin) but Signalling proteins need to be produced in response to stimuli
What is a key difference between housekeeping proteins and signalling proteins?
House keeping proteins have a long half life (may be able to last the cells life time) while signalling proteins have short half lives (may only be a few seconds)
What causes a signalling protein to be produced?
An intERcellular signal (e.g. a hormone attaching to ligands binding surface receptors) will be transduced to cytoplasm then amplified by a cascade of intRAcellular signals. This activates gene transcription temporarily for the produced of a signalling protein
Why do you not want signalling proteins to be produced all the time?
They may disturb the normal function of the cell
What strand of DNA does the RNA copy off?
The template strand (3’ -> 5’)
What is transcription?
The synthesis of RNA from a DNA template
What is RNA a copy of?
The non-template/coding strand
What is a difference in the formation of RNA from DNA?
Hydrogen bonds form between complementary base pairs (between mRNA and template strand) THEN the phosphodiester bond is formed by RNA polymerase (forms backbone of the mRNA)
What is a major difference between transcription between eukaryotic and prokaryotic cells?
The speed of response (prokaryotes can respond to an instant signal quicker to stimuli), eukaryotic cells have much greater control of the transcription process
What initiates the transcription process?
Basal transcription factors bind to appropriate place on the promoter region of the DNA
How do basal transcription factors initiate transcription?
Match RNA polymerase with appropriate promoter region in DNA
How many types of RNA polymerase do eukaryotic cells have? What functions do they each do differently?
There are 3 types (I, II, III) Each transcribes a different class of RNA
What can RNA polymerase II do unique from the other ones
It is the only one that can transcribe genes that code for proteins therefore producing mRNA
What is a key difference between eukaryotic and prokaryotic transcription promoter region location?
Prokaryotic promoter regions are generally just in front of the gene while eukaryotic promoters may be several hundred bases away
What do transcription factors do to the DNA? What does this allow
Causes melting which causes the two strands to divide
This allows the RNA polymerase to attach onto the template strand
What is a distal enhancer?
A section of the DNA which mediator proteins attach to so the bending protein enables the bending protein to attach onto a group of increasing the rate of transcription
Are distal enhancers easy to find? Where could you find them?
No, they are generally found very far away from the gene and either up or down stream in intergenic regions (non-coding regions)
What are the 3 step of eukaryotic transcription initiation?
Initiation, elongation, termination
What are the steps involved in transcription initiation?
Transcription factors bind to promotoer, in particular the TATA box.
RNA polymerase II then binds the transcription factors and initiation codon from DNA
RNA polymerase II moves along 3’-5’ manufacturing mRNA ni 5’-3’ direction
What is transcription elongation?
The process of unwinding the DNA to access the template strand
How does the DNA get unwound?
The RNA polymerase II moves along the DNA
How much of the DNA does the RNA polymerase II unwind at a time?
10-20 base pairs at a time
What is transcription annealing?
The process of rejoining the DNA back together after being separated during transcription elongation
Does transcription elongation annealing occur quickly after transcription? why/why not?
~40 nIt occurs quickly
If the DNA helix is separated for too long then it will be very hard for it to be rejoined back together
What is the rate of transcription in eukaryotic cells?
~40 nucleotides per second
What does transcription produce?
A mRNA strand
Does the mRNA strand contain the complete information for protein synthesis? Explain
Yes and it also includes sequence that are not translated onto proteins, these include regulatory sequences
What stops the transcription process? What happens in this?
A stop codon
mRNA detaches from the DNA and RNA polymerase
Once the mRNA sequence is created, what needs to be done to it?
It needs to be protected
What is used to protect the mRNA?
A 5’ cap modified guanine residue
What does the 5’ cap do?
protects form degradation, other RNA’s, stabilise the transcript, allow for it to move out of the nucleus and orientates it aid translation
What is the structure of a mRNA?
5’ cap, 5’ UTR (helps regulate translation), start codon, protein coding segment, stop codon (normally multiple stop codons to ensure translation stops), 3’ UTR (includes polyadenylation signal), poly-A tail
What is the function of the poly-A tail? What does the length of it determine? What happens if it gets too short?
Increases the stability of the mRNA
The longer it is the longer it lasts inside the cytoplasm
If it is too short then it will not be transcribed
What is the original mRNA strand called when straight from the transcription process? What must be done to the mRNA before it can be translated?
Primary RNA transcript
The noncoding regions must be spliced out
What are the coding and noncoding parts of a mRNA called? What are their functions?
Exons = coding regions, form final mRNA strand Introns = noncoding regions, spliced out but still important for regulatory functions
How do introns impact protein synthesis? Explain
From one piece of mRNA you can make multiple proteins
By skipping exons (due to the introns) you can make multiple iso forms of the same protein
What are the disadvantages of iso form proteins?
Can create a slightly dysfunctional protein
How does the spliced transcript compare to the non-spliced transcript of mRNA?
Spliced transcript is much shorter
How do mutations (and what kind in particular) around the exon and intron joining points impact humans?
Can causes cancers from the inappropriate splicing of mRNA
How are introns removed from the mRNA?
Splicesome protein
What is the structure of the splicesome?
Small nuclear RNA and proteins from small nuclear ribonuclear proteins (snRNPs), several of these form a splicesome
Are RNA molecules valuable to the cell? Why?
No because they can easily be made
What kind of cells do not produce many dysfunctional RNA? What is a key characteristic of them?
Stem cells, they don’t divide very fast
How does splicing work?
snRNA sequence is complementary to intron splice sites and the two bind, the intron is excised (cut out) and degraded and exons are joined, the spliceosome catalyses the reaction
What does the final spliced mRNA look like?
5’ cap, 5’UTR, coding segment, 3’UTR, poly-A tail
What is translation?
The synthesis of protein
What are the three stages of translation?
Initiation, elongation and termination
What is the function of tRNA?
Its is an adapter molecule that holds amino acids and interacts with the mRNA codons so the amino acids are ordered according to the mRNA
Why are tRNA molecule necessary?
The amino acids can’t physically fit onto the mRNA chain and require an adapter molecule
Are tRNA sparse? Where are they found?
No, they are highly abundant and found floating throughout the cytoplasm
Does aminoacyl tRNA synthetase vary much between different species?
No, it is highly conserved and although there are two variations found among species they perform the same function
How are amino acids attached onto the tRNA? What input does it need to work? What kind of bond does it create on the tRNA?
Enzymes call aminacyl tRNA synthetases ‘charge’ the tRNA by catalysing the addition of amino acids onto tRNA
Requires ATP
Creates a strong covalent bond
What are the steps of how amino acids are loaded onto tRNA’s?
1 - aminoacyl tRNA synthetase binds ATP and amino acid (on its active site FYI)
2 - Reaction leaves AMP and amino acid bound to tRNA synthetase and produces two phosphate groups, activated amino acid has high energy potential
3 - activated amino acid transferred onto tRNA which docks into tRNA synthetase specific to that amino acid, produces an aminoacyl tRNA
4 - aminacyl tRNA then released to be used in translation
What is AMP?
A derivative of ATP that is used in used in signalling
How many types of of aminoacyl tRNA synthetase are there? Explain
20, one for each amino acid
What is the shape of the tRNA? Is this a textbook or real shape?
Have a cloverleaf secondary structure
How it is described in text books
Describe the bonds in the structure of the tRNA
Stems are created by hydrogen bonding
Pair bonding along the stems
What is the structure of tRNA
Anti-codon at the base
3’ prime end at the top for binding with amino acids
Loops of unpaired bases
What do the loops of unpaired bases and pair bonding determine?
What kind of aminoayl tRNA synthatase is used to join amino acid to tRNA
Are all tRNA’s the same?
No, they vary greatly - loops can be of different sizes, non-conventional bases differ, anti-codon is specific to the amino acid being carrier
What is the real life shape of the tRNA?
An L-shaped tertiary structure
What is a function of the L-shaped tertiary structure of the tRNA?
The amino acid is kept far away from the mRNA strand protecting it and allowing for the amino acids to bond together to form a peptide chain
How many codons are there and how many types of tRNA are there?
61 codons but there are only 40 types of tRNA
What theory explains how only 40 tRNA pair up with 61 different codons?
Can be explained by the wobble hypothesis
What is the wobble hypothesis?
The theory where tRNA anti-codons can bond to a codon whose third position requires a nonstandard base pairing therefore creating more combinations
eg. ACG on anticodon can bond onto UGC or UGU on codon
Where are ribosomes made?
They are assembled in the nucleolus and transported to the cytosol
What are the two forms of amino acids?
Non-ionised and ionised
What does the ionisation of amino acids allows? Where does the ionisation of occur
It allows for amino acids to be joined together, occurs on the carboxyl group (e.g. —OH turns into —OH^- )
What are the functions of amino acids? How many types of amino acids are there
They are the building block molecules that form proteins, 20
What are the impacts on the properties and functions of proteins by amino acids?
Size of the protein/how many amino acids are there
Content of the amino acids
Sequence of amino acids
How does the size of the protein impact its function
Smaller proteins (e.g. hormones) are short peptides = signalling proteins which can move freely Larger proteins are very complicated globular = stay within the cell
How is the order of amino acids in a protein indicated?
Indicated by the sequence of amino acids from the positive end to the negative end of the polypeptide chain
What causes the charged ends on a polypeptide chain? What are they called?
Positive end created by H3N+ and is called N-terminus
Negative end created by COO- and is called C-terminus
How does the ribosome bind together amino acids?
Carboxyl group (COO-) is covalently bonded onto the amino group (H3N+) (produces H2O as waste product)
How do the side chain appear on a polypeptide chain?
They stick out from the back bone
How rigid is the polypeptide chain? What can impact this rigidity?
It is very flexible, secondary bonds make it more rigid
What is the criteria for a peptide vs protein in the number of amino acids?
50> = peptide
50 + = protein
Does a protein have to be a continuous length of 50+ amino acids? Explain
No, can be multiple polypeptides bonded together
What are ribosomes made of?
Both protein and rRNA (ribosomal RNA)
What are the components of the ribosome? What do these components do?
2 sub units, large + small
Large = forms peptide bond
small = hold mRNA in place
What are the 3 sites where tRNA can be found on a ribosome during translation?
A site, P site and E site
What does the A site do?
Acceptor site of aminoacyl tRNA and binds to mRNA codon
What does the P site do?
Where the peptide bond forms adding amino acid to the polypeptide chain
What does the E site do? Why is it important to have this compartment?
Exit site for the tRNA
So that the tRNA doesn’t interact with the amino acid again
Is the mechanism for which tRNA anticodons recognise codons controlled? How does this work?
No, it is a matter of trial and error, the tRNA will attempt to get into the acceptor site and hope to bond onto the codon
How does the bonding process onto the codon work?
tRNA will go into A site of ribosome, it will then move around trying to match anti codon and codon. If it can’t find the codon then it will pop straight back out otherwise it continues to the P site
What is the process of translation initiation trying to achieve?
To bind the small ribosomal subunit onto the large ribosomal sub unit
What are the step in translation initiation?
1 - small ribosomal sub unit binds the aminoacyl tRNA carrying modified start codon, formyl methionine
2 - small ribosomal subunit and tRNA bind the 5’ cap of mRNA on mRNA binding site
3 - small ribosomal subunit looks down from tRNA of formyl methionine to find start codon
4 - large ribosomal subunit then bonds onto small subunit
What is the difference between how the formyl methionine binds to the small sub unit vs all other tRNA?
Formyl goes straight into the P site while other tRNA must go through the A site first
What is translation elongation?
?It is the step where the amino acids are being joined together to form a polypeptide
What are the steps in translation elongation?
1 - tRNA anti codon bonds onto the codon of mRNA at the A site –> GTP is used to increase accuracy and efficiency of this process
2 - large ribosomal sub unit catalyses formation of the peptide bond between the amino acid on the P site with the amino acid on the A site from the C-terminus of the polypeptide chain
3 - polypeptide chain is transferred to the tRNA on the A site as the tRNA on the P site moves onto the E site
4 - tRNA which is attached to polypeptide chain moves into the P site then repeat (movement between site energised by GTP FYI)
What is GTP?
Energy source used by ribosomes during translation
What is the purpose of translation termination?
To stop producing a polypeptide chain and to release the polypeptide chain
What are the steps in translation termination?
1 - A release factor binds to the stop codon (does not include a tRNA molecule as there is no amino acid to attach FYI)
2 - Release factor include a water molecule promoting hydrolysis of bond between polypeptide and tRNA at P site
3 - polypeptide is released and the small ribosomal subunit disassociates with the large subunit, this uses 2 more GTP molecules
